Many tumor cells depend on the activity of tyrosine kinases, which act, among other functions, to depress apoptosis in the cell. The tyrosine kinases are usually overproduced in malignant cells, which contributes to the cell's ability to resist apoptosis. Modulating the activity of these proteins provides an effective means of treating cancer while not unduly damaging normal tissues. For example, about 25% of breast tumors express unusually high levels of the Her2 protein, a tyrosine kinase receptor that normally plays a part in the development of the mammary epithelium. Herceptin® (Trastuzumab) is a humanized antibody that is currently used to treat breast cancer by targeting and blocking the function of the Her2 protein. Other treatments focus on interfering with the receptors to overexpressed tyrosine kinase proteins. Receptors include HER2/neu and IGF-1R. See, Meric et al. (April 2002) J. Am. Coll. Surg. 194(4):488-501.
The major lignin in chaparral, known as nordihydroguaiaretic acid (NDGA) is a potent antioxidant and was originally used in commercial food products as a preservative. See, U.S. Pat. No. 2,644,822. Later, it was discovered that NDGA is useful in the treatment of diabetes. Hsu et al. (2001) Cell Transplant. 10(3):255-262. More recently, NDGA was investigated as a treatment for cancer because it inhibits the platelet derived growth factor receptor and the protein kinase C intracellular signalling family, which both play an important role in proliferation and survival of cancers. Moreover, NDGA induces apoptosis in tumor xenografts. Although it is likely to have several targets of action, NDGA is well tolerated in animals. However, high concentrations of NDGA are required for efficacy and it has been suggested that more potent analogs may be required. See, McDonald et al. (2001) Anticancer Drug Des. 16(6):261-270.
Other cancer drugs include doxorubicin hydrochloride (DOX), which is used alone or in combination with other drugs for treatment of malignant lymphomas and leukemias. DOX is believed to bind DNA and inhibit nucleic acid synthesis. Examples of tumors amenable to treatment with DOX are acute lymphoblastic leukemia, acute myeloblastic leukemia, Wilm's tumor, soft tissue and bone sarcomas, breast carcinoma and ovarian carcinoma. The dosage needs to be closely monitored because it can cause irreversible cardiac damage. A typical dose for adults, when given intravenously is 60-75 mg/m2 once in 21 days, or 30 mg/m2 daily for 3 days every four weeks, where the total cumulative dose should not exceed 550 mg/m2 without monitoring for cardiac function.
It is well established that breast cancer is regulated by receptors for the female sex steroids, estrogen and progesterone. It is now appreciated that receptor tyrosine kinases (RTKs) are also very important for breast cancer growth (Arteaga CL, Moulder SL, Yakes FM: HER (erbB) tyrosine kinase inhibitors in the treatment of breast cancer. Semin Oncol 29:4-10, 2002; Averbuch S, Kcenler M, Molis C, Wakeling A: Therapeutic potential of tyrosine kinase inhibitors in breast cancer. Cancer Invest 21:782-791, 2003; Baserga R: The IGF-I receptor in cancer research. Exp Cell Res 253:1-6, 1999; Dickson RB, Lippman ME: Growth factors in breast cancer. Endocr Rev 16:559-589, 1995; Gross JM, Yee D: The type-1 insulin-like growth factor receptor tyrosine kinase and breast cancer: biology and therapeutic relevance. Cancer Metastasis Rev 22:327-336, 2003; and Nahta R, Hortobagyi GN, Esteva FJ: Growth factor receptors in breast cancer: potential for therapeutic intervention. Oncologist 8:5-17, 2003).
Accordingly, RTKs are targets for anti-tumor therapy. RTKs are transmembrane proteins that typically contain an extracellular ligand binding domain, activated by peptide hormones, and an intracellular tyrosine kinase domain. Two RTKs of demonstrated importance in breast and other cancers are the insulin-like growth factor receptor (IGF-1R) (Heinemann V: Present and future treatment of pancreatic cancer. Semin Oncol 29:23-31, 2002) and c-erbB2/HER2/neu (HER2/neu) (Morin MJ: From oncogene to drug: development of small molecule tyrosine kinase inhibitors as anti-tumor and anti-angiogenic agents. Oncogene 19:6574-6583, 2000). Based on their major role in regulating cancer cell growth and survival, inhibitors of these RTKs are undergoing drug development (Morin MJ: From oncogene to drug: development of small molecule tyrosine kinase inhibitors as anti-tumor and anti-angiogenic agents. Oncogene 19:6574-6583, 2000; Bruns CJ, Solorzano CC, Harbison MT, Ozawa S, Tsan R. Fan D, Abbruzzese J, Traxler P, Buchdunger E, Radinsky R, Fidler IJ: Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma. Cancer Res 60:2926-2935, 2000; Bruns CJ, Harbison MT, Davis DW, Portera CA, Tsan R, McConkey DJ, Evans DB, Abbruzzese JL, Hicklin DJ, Radinsky R: Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms. Clin Cancer Res 6:1936-1948, 2000; Blum G, Gazit A, Levitzki A: Substrate competitive inhibitors of IGF-1 receptor kinase. Biochemistry 39:15705-15712, 2000).
Signaling via the IGF-1R is important for normal cell growth and differentiation. In addition, the IGF-1R stimulates mitogenesis and suppresses apoptosis of cancer cells (Lowe WL: Biological actions of the insulin-like growth factors. In LeRoith D (ed): Insulin-like growth factors: molecular and cellular aspects. Boca Raton, CRC Press, 1991). Following binding of the ligand to the IGF-1R, a conformational change induces trans-autophosphorylation of the β-subunits on select tyrosine residues, and subsequent activation of tyrosine kinase activity (Lowe WL: Biological actions of the insulin-like growth factors. In LeRoith D (ed): Insulin-like growth factors: molecular and cellular aspects. Boca Raton, CRC Press, 1991). Phosphorylation of several target substrates activates divergent signaling cascades, though the anti-apoptotic effects of the IGF-1R are primarily mediated via the Akt/PKB pathway (Kulik G, Klippel A, Weber MJ: Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 17:1595-1606, 1997).
Tyrosine phosphorylation of the insulin receptor substrate (IRS) family of proteins by the IGF-1R allows binding of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K) to the IRS proteins via SH2 domains. Activated PI3K serine phosphorylates and activates the serine kinase Akt (Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA: Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15:6541-6551, 1996). Akt can phosphorylate the protein BAD, which prevents BAD from forming a pro-apoptotic complex with Bcl-2 proteins (Virdee K, Parone PA, Tolkovsky AM: Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival. Curr. Biol 10:1151-1154. 2000).
Interruption of the IGF-1R signaling system, either by reducing effective IGF-1 levels or targeting the receptor., can block growth and proliferation of cancer cells (Kahan Z. Varga JL, Schally AV, Rekasi Z, Armatis P, Chatzistamou L, Czompoly T, Halmos G: Antagonists of growth hormone-releasing hormone arrest the growth of MDA-MB-468 estrogen-independent human breast cancers in nude mice. Breast Cancer Res Treat 60:71-79, 2000: Neuenschwander S, Roberts CT, Jr., LeRoith D: Growth inhibition of MCF-7 breast cancer cells by stable expression of an insulin-like growth factor I receptor antisense ribonucleic acid. Endocrinology 136:4298-4303, 1995: Prager D, Li HL. Asa S, Melmed S: Dominant negative inhibition of tumorigenesis in vivo by human insulin-like growth factor I receptor mutant. Proc Natl Acad Sci USA 91:2181-2185, 1994; Weckbecker G, Tolcsvai L, Liu R, Bruns C: Preclinical studies on the anticancer activity of the somatostatin analogue octreotide (SMS 201-995). Metabolism 41:99-103, 1992; and Yee D, Jackson JG, Kozelsky TW, Figueroa JA: Insulin-like growth factor binding protein 1 expression inhibits insulin-like growth factor I action in MCF-7 breast cancer cells. Cell Growth Differ 5:73-77. 1994). While overexpression of the IGF-1R can drive transformation and mitogenesis, it is the requirement for its constitutive presence in cancer cells (Rubin R, Baserga R: Insulin-like growth factor-I receptor. Its role in cell proliferation, apoptosis, and tumorigenicity. Lab Invest 73:311-331, 1995) that makes this RTK an attractive target for anti-tumor therapies.
The HER2/neu (c-erbB-2) protooncogene encodes a 1,255 amino acid, 185 kDa member of the class I RTK family. HER2/neu is overexpressed in 20-30% of breast cancers, most commonly via gene amplification, and overexpression is associated with poor prognosis in these patients (Slamon DJ, Godolphin W. Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J. Ullrich A,.: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707-712, 1989; Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177-182, 1987). Evidence from transgenic animal studies indicates that HER2/neu overexpression directly contributes to transformation and tumor progression (Bol D, Kiguchi K, Beltran L, Rupp T, Moats S, Gimenez-Conti I, Jorcano J, DiGiovanni J: Severe follicular hyperplasia and spontaneous papilloma formation in transgenic mice expressing the neu oncogene under the control of the bovine keratin 5 promoter. Mol Carcinog 21:2-12, 1998; Bouchard L, Lamarre L, Tremblay PJ, Jolicoeur P: Stochastic appearance of mammary tumors in transgenic mice carrying the MMTV/c-neu oncogene. Cell 57:931-936, 1989; and Lucchini F, Sacco MG, Hu N, Villa A, Brown J, Cesano L, Mangiarini L, Rindi G, Kindl S, Sessa F,.: Early and multifocal tumors in breast, salivary, harderian and epididymal tissues developed in MMTY-Neu transgenic mice. Cancer Lett 64:203-209, 1992), and suggests that its prognostic significance arises from the particularly aggressive phenotype it confers (Hynes NE, Stern DF: The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta 1198:165-184, 1994). The efficacy of targeting HER2/neu in anti-cancer therapy has been demonstrated by the clinical use of an antibody to HER2/neu to treat certain patients with breast cancer (Albanell J, Baselga J: Trastuzumab, a humanized anti-HER2 monoclonal antibody, for the treatment of breast cancer. Drugs Today (Barc) 35:931-946, 1999).
Nordihydroguaiaretic acid (NDGA) is a phenolic compound that was identified as a major component of a tea made from resinous extracts of the creosote bush Larrea divaricatta. It has been used for centuries by Native North Americans as a remedy for diverse illnesses, including tumors (Duisberg PC: Desert Plant Utilization. Texas J Sci 4:269, 1952: Hawthorn P: Medicinal uses of plants of Nevada used by Indians. Contr Flora Nevada 45:1-139, 1957). NDGA has been reported to inhibit the growth of various tumors both in vitro and in animals (Wilson DE, DiGianfilippo A, Ondrey FG, Anderson KM, Harris JE: Effect of nordihydroguaiaretic acid on cultured rat and human glioma cell proliferation. J Neurosurg 71:551-557, 1989; Avis IM, Jett M, Boyle T, Vos MD, Moody T, Treston AM, Martinez A, Mulshine JL: Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J Clin Invest 97:806-813, 1996; Rose DP, Connolly JM: Effects of fatty acids and inhibitors of eicosanoid synthesis on the growth of a human breast cancer cell line in culture. Cancer Res 50:7139-7144, 1990: and Shimakura S, Boland CR: Eicosanoid production by the human gastric cancer cell line AGS and its relation to cell growth. Cancer Res 52:1744-1749, 1992). NDGA also has been reported to induce apoptosis in a variety of cell lines (Ding XZ, Kuszynski CA, El Metwally TH, Adrian TE: Lipoxygenase inhibition induced apoptosis, morphological changes, and carbonic anhydrase expression in human pancreatic cancer cells. Biochem Biophys Res Commun 266:392-399, 1999: La E, Kern JC, Atarod EB, Kehrer JP: Fatty acid release and oxidation are factors in lipoxygenase inhibitor-induced apoptosis. Toxicol Lett 138:193-203, 2003; Seufferlein T, Seckl MJ, Schwarz E, Beil M, Wichert G, Baust H, Luhrs H, Schmid RM, Adler G: Mechanisms of nordihydroguaiaretic acid-induced growth inhibition and apoptosis in human cancer cells. Br J Cancer 86:1188-1196, 2002; Tong WG, Ding XZ, Witt RC, Adrian TE: Lipoxygenase inhibitors attenuate growth of human pancreatic cancer xenografts and induce apoptosis through the mitochondrial pathway. Mol Cancer Ther 1:929-935, 2002; and Tong WG, Ding XZ, Adrian TE: The mechanisms of lipoxygenase inhibitor-induced apoptosis in human breast cancer cells. Biochem Biophys Res Commun 296:942-948, 2002). Still, the mechanism of this anti-cancer effect of NDGA is not well understood. It has been reported that NDGA inhibits the tyrosine kinase activity of the platelet-derived growth factor receptor (PDGFR), but not the epidermal growth factor receptor (EGFR), in cells and in vitro (Domin J, Higgins T, Rozengurt E: Preferential inhibition of platelet-derived growth factor-stimulated DNA synthesis and protein tyrosine phosphorylation by nordihydroguaiaretic acid. J Biol Chem 269:8260-8267, 1994). While one report suggests that NDGA is inactive against the IGF-1R (Seufferlein T, Seckl MJ, Schwarz E, Beil M, Wichert G, Baust H. Luhrs H, Schmid RM, Adler G: Mechanisms of nordihydroguaiaretic acid-induced growth inhibition and apoptosis in human cancer cells. Br J Cancer 86:1188-1196, 2002), a compound with a very high degree of structural homology to NDGA has been described as a potent inhibitor of this receptor(Blum G. Gazit A, Levitzki A: Substrate competitive inhibitors of IGF-1 receptor kinase. Biochemistry 39:15705-15712, 2000: Blum G. Gazit A, Levitzki A: Development of new insulin-like growth factor-1 receptor kinase inhibitors using catechol mimics. J Biol Chem 278:40442-40454, 2003). The effects of NDGA on the HER2/neu receptor, which also plays a critical role in breast cancer, have not been explored. We have now found that NDGA antagonizes the activation of both the IGF-1 and HER2/neu receptors inhibits the cellular anti-apoptotic signaling pathway of the IGF-1R, and inhibits the growth of breast cancer cells both in vitro and in vivo.
There is a need for therapeutic cancer treatments that block the tyrosine kinase receptors with lower dosages of these powerful drugs to reduce side effects. The present invention addresses this and other related needs.